NON-HUMAN TRANSGENIC ANIMAL EXPRESSING CA-AHR

Description

[0001] The present invention relates to a mutant construct for a constitutively active aryl hydrocarbon receptor (CA-AhR), a transgenic non-human animal expressing CA-AhR, and an animal model for the study of the molecular mechanisms of cancer, in particular stomach cancer. The invention also relates to methods of screening and/or investigating carcinogenic and anti-carcinogenic compounds, screening and/or investigating drug candidates, as well as compounds discovered or developed using this method.

Background of the invention

[0002] The dioxin / aryl hydrocarbon receptor (AhR) belongs to a specific class of transcription factors, basic helix-loop-helix/Per-Arnt-Sim domain (bHLH/PAS) proteins, which is emerging as an important battery of regulatory factors seemingly designed to respond to environmental cues. Other members of this family include the hypoxia-inducible factor HIF-1α, the rhythmicity regulatory protein Clock, the neuro-regulatory protein Sim, and Arnt, an essential partner factor for all of the factors mentioned above including the AhR (1). Arnt is recruited to the AhR in a ligand-dependent manner to facilitate recognition of xenobiotic response elements of target promoters.

[0003] The ligand-activated AhR mediates transcriptional activation of a network of genes encoding enzymes such as CYP1A1, CYP1A2, glutathione S-transferase Ya, UDP-glucuronosyl-transferase 1A6 and NAD(P)H quinone oxidoreductase-1 that function in the oxidative metabolism of xenobiotics (2). Well-characterized ligands of the AhR are polycyclic aromatic hydrocarbons formed during combustion processes and polychlorinated dioxins and coplanar biphenyls that contaminate industrial chemicals and the environment (2). Thus, AhR-mediated signalling pathways provide a first line of defence against potentially toxic environmental pollutants. On the other hand, induction of oxidative metabolic processes by the AhR can also cause the production of highly carcinogenic metabolites, creating a strong link between AhR activation and chemical carcinogenesis (3). In addition, the receptor appears to mediate by as yet unclear mechanisms a wide range of toxic effects by chlorinated dioxins including birth defects, impaired reproductive capacity, and immune suppression (1). A number of independent loss-of-function studies performed by gene disruption in mice have not yielded conclusive information with regard to a possible developmental role of the receptor (4-7). In view of its critical role in mediating metabolic responses to environmental pollutants, the sole biological function of the AhR could therefore be restricted to regulation of adaptive responses to xenobiotics. This notion seems to be corroborated by the fact that a putative physiological function of the AhR remains to be determined. Against this background the present inventors have performed a gain-of-function study to examine possible biological functions of the AhR system. To this end, a constitutively active AhR mutant (CA-AhR) was created and expressed in transgenic mice to study possible AhR-mediated biological effects that are generated in the absence of any exposure to environmental contaminants.

Prior art

[0004] U.S. 5,378,822 discloses recombinant DNA molecules which encode murine and human Ah receptors, which are used to generate large quantities of Ah-receptor protein for use in competitive binding assays used for detecting environmental pollutants or for regulating gene expression in response to receptor agonists. Another use is for the generation of recombinant organisms that can serve as biomonitors for environmental pollutants, or for detecting human and wildlife populations that have high susceptibility to environmental pollutants.

[0005] US 5 650 283 discloses mammalian cells constitutively expressing AhR for the study of pollutants. It is suggested therein to truncate AhR in the ligand binding region. The document "Breast Cancer Research and Treatment", vol. 63 2000, pages 117-131 discusses the involvement of constitutive expression of AhR in tumour cell growth.

Summary of the invention

[0006] The present invention makes available a powerful tool for the study of cancer, based on a novel expression construct for a constitutively active hydrocarbon receptor CA-AhR. The invention further comprises transgenic non-human animals, preferably mammals, expressing CA-AhR in one or more tissues thereof. An animal model based on said transgenic non-human animals forms the basis for novel methods e.g. for the study of cancer; for the screening of compounds, such as drug candidates; for the investigation of the molecular mechanisms of cancer, in particular stomach cancer; for the investigation of the mechanisms of highly differentiated adenocarcinoma etc. Likewise, an in vitro model based on transformed cells or cell lines, functionally incorporating the inventive construct are disclosed.

[0007] The invention will be further defined in the description, examples and attached claims, hereby incorporated in their entirety.

Brief description of the drawings

[0008] The invention will be described in closer detail in the description and examples below, with reference to the attached drawings, in which

Figure 1 shows the constitutive activity of CA-AhR. (A) Schematic representation of the wild type mouse AhR (mAhR) and of CA-AhR. (B) Functional activity of CA-AhR in CHO cells. Cells were transiently transfected with an AhR-dependent luciferase reporter gene, and expression vectors encoding Amt, wild type AhR, or CA-AhR. The control lanes (Ctrl) represent activity from the reporter gene alone and empty expression plasmid. Data are from one experiment performed in duplicate and are representative of at least three independent experiments. (C) Detection of the AhR and CA-AhR proteins expressed following transient transfection of CHO cells. Whole cell extracts were analyzed by immunoblotting using anti-AhR antibodies. The star indicates non-specific immunoreactivity. (D) Expression and functional activity of CA-AhR in 8 month old female mice. RNA blot analysis (2µg poly-A RNA) showing expression of the endogenous AhR, CA-AhR and the target genes CYP1A1 and CYP1A2. The expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is shown as RNA loading control of corresponding tissues.

Figure 2 shows the functional activity of CA-AhR in the mouse thymus and mortality time course. (A) RNA blot (30 µg total RNA) showing expression of CYP1A1 mRNA in thymus of six month old wild type versus age-matched heterozygous and homozygous CA-AhR mice, treated with vehicle (corn oil) or TCDD as indicated. (B) The relative thymus weight (g/g body weight) was decreased in homozygous CA-AhR animals up to six months of age. Closed bars represent wild type and open bars represent CA-AhR animals. At least four female animals of each genotype and age were examined. The star indicates p<0.05, as assessed by two-tailed Students t-test. (C) Ages of the homozygous CA-AhR mice found dead stratified by sex (closed symbols for males, open for females) and strain (triangles for strain "A3" and circles for strain "Y8").

Figure 3 shows how striking neoplastic lesions are observed in the stomach. (A) Normal stomach from a 12 month old wild type male showing the forestomach (fs) and the glandular stomach (gs). (B) At 3-4 months of age single small cysts close to the limiting ridge were seen in CA-AhR mice (arrow). (C) In older CA-AhR animals (6-12 months) the cystic tumours were more numerous and occupied a larger area of the stomach. (D) In the most severe cases (9-12 months of age), the stomach was adherent to adjacent organs such as spleen (sp), pancreas (panc) and liver (liv). (E) Normal stomach from a 6 month old wild type male mouse showing the muscularis propria layer (mp) and the limiting ridge (lr) constituting the border between the squamous epithelium of the forestomach (fs) and the glandular epithelium (ge; Hematoxylin and Eosin staining [HE], bar=0.5 mm). (F) Close to the limiting ridge a rupture of the submucosa by neoplastic crypts is seen in a 3.5 month old CA-AhR male. Note glands within the stroma of the limiting ridge (HE, bat=0.5 mm). (G) Larger magnification of boxed area in Fig. 3F (HE, bar=0.15 mm). (H) Stomach from a 12 month old CA-AhR male with severely distorted tissue architecture (HE, bar=1.25 mm). (I) Glands underlying the serosa (ser) in a 12 month old CA-AhR female with characteristics of a hamartoma (ham), i.e. a defined structure containing lymphatic tissue, vessels and fat (HE, bar=0.5 mm). Note also invasion (arrow) of glands from the glandular epithelium (ge) into the muscularis propria (mp).

Figure 4 shows intestinal metaplasia, adherence to adjacent organs and expression of CA-AhR in the gastrointestinal tract. (A, B) Glandular structures located in the muscularis propria with cells resembling foveolar epithelium (fe) and pyloric glands (pg) showing intestinal metaplasia in a 9 month old CA-AhR male. Stainings: Hematoxylin and Eosin (HE; panel A) and Alcian Blue pH 2.5 (panel B). Bars=0.1 mm. (C, D) Invading crypts surrounded by connective tissue (ct) invade the submucosa (sm) by penetrating through the muscularis mucosa (mm), submucosa (sm) layers and into the muscularis propria (mp) in a 9 month old CA-AhR female. Stainings: HE (panel C) and van Gieson (panel D). Bars=0.1 mm. (E, F) Squamous cysts on the caecum showing colonic glands (cg) and squamous epithelium (sq.e) of a 9 month old CA-AhR male (HE, bar=0.5 mm). (G) The expression and activity of CA-AhR in the alimentary tract is highest in the glandular stomach. RNA blot (2 µg poly-A RNA) showing expression of CA-AhR, endogenous AhR (AhR), CYP1A1 and GAPDH mRNA in different parts of the alimentary tract of homozygous CA-AhR mice three months of age.

Description

[0009] Before the present construct, transgenic animals incorporating said construct, animal models and methods, based on the use of said animals, are disclosed and described, it is to be understood that this invention is not limited to the particular configurations, process steps, and materials disclosed herein as such configurations, process steps, and materials may vary somewhat. It is to be understood that the terminology employed herein is used for the purpose of describing particular embodiments only and is not intended to be limiting since the scope of the present invention will be limited only by the appended claims and equivalents thereof.

[0010] In the description, examples, and claims, the following abbreviations will be used: AhR = Aryl hydrocarbon (dioxin) receptor; Arnt = AhR nuclear translocator, CA-AhR = Constitutively Active AhR; CYP1A1 = Cytochrome P450 1A1; HE = Hematoxylin-Eosin; TCDD = 2,3,7,8-tetrachlorodibenzo-p-dioxin.

[0011] The present inventors have surprisingly found that expression of CA-AhR in transgenic mice induces a pronounced lethality beginning at six months of age, correlating with the development of severe tumours in the stomach. Thus, this study clearly demonstrates the oncogenic potential of the AhR. It has been difficult to unambiguously interpret the histopathology of the stomach tumours in the CA-AhR mice. The well-organised glandular structures and the low levels of cellular atypia argue for a benign phenotype. On the other hand, the reduced life span, the aggressive, expanding invasion of all stomach layers and the adherence to surrounding organs point toward a more malignant phenotype. Intestinal metaplasia was widespread in the CA-AhR tumours and this is regarded as a pre-cancerous lesion (17). Furthermore, a subgroup of human intestinal-type gastric carcinoma has recently been described where the cancer cells also are highly differentiated (18). Given the striking gastric oncogenic phenotype of the CA-AhR mice it is interesting to note that the most physiological candidates of receptor ligands are indole derivatives, most notably indolo[3,2-b]carbazole that are generated in the acidic environment of the stomach from dietary precursors, e.g. indolo-3-carbinol (19). Moreover, certain food-born heterocyclic amines that are generated during the food cooking process also constitute AhR ligands (20). Thus, the correlation between presence of putative dietary receptor ligands and a possible role of the AhR in homeostatic control of cells of the gastric mucosa presents an intriguing biological scenario that remains to be scrutinised in closer molecular detail.

[0012] Stomach cancer is the second most common human malignancy in the world (21). The role (if any) of the AhR in development of this cancer form is not known. Interestingly, stomach cancer is more commonly found in men than in women (21, 22), a sex difference that is reflected in the CA-AhR mice. Some epidemiological studies show an increased incidence of stomach cancer in human populations exposed to herbicides (23) or fatty fish (24) contaminated with TCDD or other dioxins. More commonly discussed risk factors for stomach cancer is diet containing mutagenic nitrosating compounds, as well as infection with Helicobacter pylori (22). However, the CA-AhR animals in this study received conventional rodent feed, and no infection by Helicobacter was detected by selective culture of tissue homogenates (data not shown). Given the absence of any known carcinogen, it is unlikely that induction of drug metabolising enzymes and ensuing bioactivation of mutagens can explain the oncogenic effect of the AhR. A more intriguing hypothesis is that a network of critical growth control genes is dysregulated by the CA-AhR.

[0013] In the stomach mucosa high levels of endogenous AhR mRNA are detected on gestational day 15.5 of the developing mouse (25). The present inventors have detected expression of the CA-AhR in the stomach of new-born mice (data not shown). Thus, it is very probable that the present mouse model reflects a situation of early in utero exposure to AhR ligands, which continues post-natally. Strikingly, there is a paucity of data with regard to the long-term effects following in utero exposure to dioxins and other environmental pollutants constituting AhR ligands. In addition to dioxins xenobiotic AhR ligands include halogenated biphenyls, and a large number of non-halogenated polycyclic aromatic hydrocarbons, e.g. benzo[a]pyrene, 9,10-dimethylbenz[a]anthracene, and 3-methylcholanthrene. In this context it is noteworthy that the AhR has recently been proposed to differentially regulate various target genes depending on the chemical nature of the receptor ligand (26). The AhR has been reported to activate Bax gene transcription when exposed to 9,10-dimethylbenz[a]anthracene but not when occupied with TCDD as ligand (26). Obviously, this model needs to be further substantiated. Strikingly, the present model system may provide an experimental tool to resolve this issue. Notably, the possible biological effects mediated by the activated receptor per se (i.e. produced by the CA-AhR) can be compared to the effects produced by the various ligand-stimulated receptor forms. However, this scenario needs to be further experimentally elucidated, taking into account that all classes of receptor ligands may not yet have been identified.

[0014] Interestingly, several species of laboratory animals treated with AhR ligands have been reported to develop lesions in the glandular stomach mucosa that resemble the findings in the CA-AhR mice of the present invention. For instance, adenocarcinoma is observed after injection of 3-methylcholantrene into the stomach wall of several strains of mice (27, 28). Whether caused by reactive 3-methylcholantrene metabolites or some other mechanism, a potential role for the AhR in 3-methylcholantrene-induced stomach cancer is supported by the observation that the DBA mouse strain expressing a low affinity AhR variant is resistant to developing 3-methylcholantrene-induced stomach tumours (27). In addition, hyperplasia of the gastric mucosa and cysts in the submucosa of Rhesus monkeys (29) and adenocarcinoma of rat glandular stomach (30) have been observed after exposure to dietary mixtures of polychlorinated biphenyls that have the potential to activate the AhR. Taken together, these observations indicate an important role of the AhR in gastric tumorigenesis and thus also in the control of growth and proliferation of gastric epithelial cells.

[0015] There exist seemingly contradictory reports on the role of the AhR in cell cycle control. TCDD has been reported to stimulate growth of human keratinocytes (31), and mutant cells that express no or substantially reduced levels of AhR display decreased growth rates in comparison to wild type cells (32, 33). On the other hand, TCDD has been reported to induce expression of the cyclin/cdk inhibitor p27 (Kip1) in certain cells (34). Interestingly, mice develop adenocarcinomas in the glandular stomach upon expression of viral oncoproteins binding the retinoblastoma protein Rb (35-38). Notably, the AhR has recently been reported to physically interact with Rb (39, 40) via an as yet unclear mechanism, and it remains to be established whether this effect is of any relevance for the phenotype of the CA-AhR expressing mice.

[0016] Interestingly, mice overexpressing TGFα or EGF-like viral growth factors show cystic hyperplasia, intestinal metaplasia and dysplasia in the stomach (41, 42). Moreover, TGFα mRNA expression levels are known to be induced by TCDD treatment of e.g. keratinocytes (43). However, the present inventors failed to detect any increase in TGFα mRNA levels in the glandular stomach of the CA-AhR mice (data not shown). Thus, it will now be important to identify the network of genes that is dysregulated upon expression of the CA-AhR and to thereby understand a possible physiological role of the AhR in gastric homeostasis.

[0017] In conclusion, the present inventors have demonstrated that CA-AhR induces development of highly invasive stomach tumours in the absence of exposure to any known carcinogen. This study provides for the first time evidence of the direct oncogenic potential of the AhR and suggests a possible physiological role of the AhR in homeostatic control of cells of the gastric mucosa.

[0018] Consequently, the present invention makes available an expression construct for a constitutively active hydrocarbon receptor (CA-AhR), and in particular a mutant construct.

[0019] According to one embodiment of the invention, the construct is lacking a portion of the ligand binding domain. According to a particular embodiment, presently preferred by the inventors, said construct comprises a mouse AhR sequence lacking amino acids 288 - 421.

[0020] The invention further makes available a transgenic non-human animal expressing CA-AhR in one or more tissues thereof or a transgenic non-human animal functionally incorporating an expression construct as defined above.

[0021] The transgenic non-human animal according to the invention is preferably selected from the group consisting of mice, rats, moneys, sheep and rabbits.

[0022] The present invention also encompasses an isolated cell of the inventive transgenic non-human animal as defined above. The invention also encompasses an isolated cell line derived from the transgenic animal as defined above. According to one embodiment, the cell or cells is/are selected from a germ cell or a somatic cell.

[0023] The present invention makes available an animal model for the study of cancer, comprising a transgenic non-human animal comprising an expression construct for a constitutively active hydrocarbon receptor (CA-AhR) in at least one of its cells. The transgenic animal according to the invention is preferably selected from the group consisting of mice, rats, monkeys, sheep and rabbits.

[0024] The present invention also makes available an in vitro model for the study of cancer, comprising a cell having comprising an expression construct for a constitutively active hydrocarbon receptor (CA-AhR) functionally incorporated. The invention further makes available an in vitro model for the study of cancer, comprising a cell line, the cells of which comprising an expression construct for a constitutively active hydrocarbon receptor (CA-AhR) functionally incorporated.

[0025] An important embodiment of the present invention is a method for the screening of drug candidates, wherein the anti-carcinogenic effect of said drug candidates is assessed in a non-human transgenic animal or a cell or cell line thereof expressing a constitutively active hydrocarbon receptor (CA-AhR).

[0026] Another embodiment is a method for the screening of drug candidates, wherein the anti-carcinogenic effect of said drug candidates is assessed in an animal model, or in an in vitro model as defined above.

[0027] Another embodiment is a method for investigating the molecular mechanisms of cancer, wherein an animal model or in vitro model as defined above is used.

[0028] The invention also makes available a method for investigating the mechanisms of highly differentiated adenocarcinoma in the stomach, wherein an animal model or in vitro model as defined above is used.

[0029] The invention further makes available a method of inducing stomach cancer in a non-human animal for research purposes, wherein said animal is transformed with a construct expressing a constitutively active hydrocarbon receptor (CA-AhR). Transformed with the construct in this context means that the construct is functionally inserted, i.e. in proper reading frame and orientation, as is well understood by persons skilled in the art. Different expression vectors or systems are well known.

[0030] According to one embodiment of the present invention, the construct is injected into a fertilised egg of said non-human animal and the egg permitted to develop into an animal containing said construct in its genome.

[0031] Further, the invention makes available a method of inducing drug metabolising enzymes normally regulated by the Ah-receptor in the presence of a ligand for the study of drug metabolism by any member (-s) of said enzymes, wherein a non-human animal is transfected with a construct expressing a constitutively active hydrocarbon receptor (CA-AhR).

[0032] According to one embodiment of the above method, an in vitro method of inducing drug metabolising enzymes normally regulated by the Ah-receptor in the presence of a ligand for the study of drug metabolism by any member (-s) of said enzymes is assembled by transfecting cultured cells with a construct expressing a constitutively active hydrocarbon receptor (CA-AhR).

[0033] The present invention, by making the above practical and powerful research and screening tools available, also relates to drug candidates, prodrugs and treatment regimens identified by a process involving a method, an animal method or an in vitro method involving animals or cells functionally incorporating a construct expressing a constitutively active hydrocarbon receptor (CA-AhR).

[0034] In the context of drug-developments and the understanding of the molecular mechanisms of cancer, other non-human animals than mice are also of interest, especially other mammals. Rodents, for example, are widely used and especially rats and mice. As the inventive construct also can be transformed into and the CA-Ah receptor expressed in other animals, the present invention also encompasses the use of the construct in such other animals, animal models and methods based thereon.

Examples

Materials and Methods

[0035] Cell Culture, Reporter Gene and Immunoblot Assays: CHO cells were transiently transfected with an XRE-containing luciferase reporter gene construct, PTXDIR, and CMV expression plasmids encoding Arnt and either the wild type mouse AhR (8) or a mouse AhR lacking a portion of the ligand binding domain (amino acids 288-421), CA-AhR (J. McGuire, K. Okamoto, M. L. Whitelaw, H. Tanaka, L. Poellinger, studies performed, manuscript in preparation). After 48 h of incubation either in the presence of 10 nM TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) or vehicle (1% DMSO) alone, luciferase activity was assayed. Whole cell extracts were prepared as previously described (8) to monitor expression of the AhR. The extracts (30 µg protein) were separated by 7.5% SDS-PAGE, transferred to nitro-cellulose membrane and relative expression levels determined by immunodetection with anti-AhR antiserum (BioMol, PA).

[0036] Mice: The CA-AhR was subcloned between the mouse IgH intron enhancer/SV40 promoter and the SV40 polyadenylation site of pEµSR (9). Transgenic mice were created by pronuclear injection of a 5.5 kb KpnI fragment encompassing the EµSR-CA-AhR construct into fertilised C57BL/6 x CBA eggs, resulting in five founder animals carrying the CA-AhR construct in the genome. Three lines were chosen for further studies and subsequently crossed into the C57BL/6 strain for two additional generations. Transgenic CA-AhR and wild type control animals were of the same mixed genetic background. Homozygosity was verified by Southern blot analysis of genomic DNA from tail biopsies. Animals were held in ventilated filter-top cages and received conventional rodent feed (RM3, Special Diet Services) and tap water ad libitum, and were exposed to a 12-hour light/dark cycle. In TCDD exposure studies, age-matched wild type and CA-AhR female mice were treated with corn oil or various doses of TCDD dissolved in corn oil and were sacrificed three days later. Animals were sacrificed by CO₂ asphyxiation followed by cervical dislocation. All animal procedures were approved by the local ethical committee.

[0037] The sex ratio of all CA-AhR animals was 216 males and 209 females, and of the homozygous CA-AhR mice 125 males and 107 females, compared to 245 males and 246 females of the wild type mice.

[0038] Wild type and homozygous CA-AbR animals were weighed once a week during the first 3 months of life. Even though individual litters differed in weight gain, no difference was observed in either sex when average weights of 5 litters of each genotype were compared (45 wild type and 44 CA-AhR animals in total).

[0039] RNA Isolation and Northern Blot Assay: Total RNA was prepared by tissue homogenisation in a guanidinium thiocyanate buffer followed by CsCl₂-gradient centrifugation (10). Poly-A RNA was isolated from total RNA using oligo-(dT)-coupled magnetic beads (Dynal AS, Oslo, Norway). Northern blot analysis was carried out according to standard methods (10). Prehybridization and hybridization was carried out at 42°C in a formamide-containing buffer (10). The filters were hybridised overnight with ³²P-labelled cDNA probes specific for the genes indicated (11). The filters were washed with 2xSSPE at room temperature, 30 minutes 2xSSPE/2% SDS at 65°C and 30 minutes 0.1xSSPE/0.1% SDS at 65°C and subsequently exposed to autoradiographic film at -70°C and Phosphorlmager analysis (FujiFilm Inc.). The PhosphorImager results were quantified using the software provided by the manufacturer.

[0040] Histopathological Analysis: Tissues were removed and fixed in 4 % buffered formaldehyde, embedded in paraffin and cut into 4 µm thick sections that were stained with Hematoxylin-Eosin, Alcian Blue pH 2.5 or van Gieson stain according to standard procedures.

Results

[0041] In analogy to nuclear hormone receptors (12), the ligand binding domain of the AhR mediates both activation of receptor function in the presence of ligand and repression of receptor function in the absence of ligand (8, 13). Partial deletion of the minimal ligand-binding domain of the AhR results in a protein, CA-AhR (Fig. 1A), that fails to bind ligand (data not shown). This truncated receptor was constitutively active with regard to reporter gene activation (Fig. 1B) in transient transfection experiments, at CA-AhR expression levels matching those of the ligand-dependent wild type AhR (Fig. 1 C).

[0042] CA-AhR was expressed in transgenic mice under the control of an SV40 promoter and the immunoglobulin heavy chain (IgH) intron enhancer (9). Mating heterozygous CA-AhR animals yielded wild type, hetero-, and homozygous mice at a normal Mendelian 1:2:1 frequency, indicating no prenatal lethality of homozygous mutants. Both heterozygous and homozygous CA-AhR mice were fertile and showed a normal sex ratio. In agreement with other studies using IgH intron enhancer-driven expression constructs (9, 14), CA-AhR mRNA expression levels were detected in thymus and spleen (Fig. 1D) and in enriched B and T cells (data not shown) as well as in a number of non-lymphoid tissues (Fig. 1D). The ligand-activated AhR regulates expression of a battery of genes encoding xenobiotic metabolising enzymes, e.g. CYP1A1 and CYP1A2 (1). With the exception of the lung, expression of CYP1A1 mRNA was not detected in untreated wild-type mice (Fig. 1D). In contrast, all tissues that showed CA-AhR transgene expression also demonstrated induced expression at various levels of CYP1A1 mRNA (Fig. 1D). However, the variation in induced expression of this target gene did not correlate with the expression levels of CA-AhR, indicating that additional tissue-specific factors are important for the regulation of CYP1A1 expression. In addition, in the liver expression of CYP1A2 mRNA was also induced by the transgene. Taken together, this demonstrates that CA-AhR is transcriptionally active and mimics the action of the ligand-activated AhR.

[0043] To assess the level of functional activity of CA-AhR, induction of CYP1A1 mRNA expression by CA-AhR in the thymus was compared to the induction response produced in wild-type mice following oral exposure to TCDD. In homozygous CA-AhR mice, the levels of CYP1A1 mRNA were comparable to those observed in wild type mice treated with a single dose of TCDD of 3 µg TCDD/kg body weight (Fig. 2A). Upon exposure to this dose of TCDD no acute toxic effects (e.g. lethality or the wasting syndrome) are seen in mouse models (15). No effect on body weight gain was observed in either male or female CA-AhR mice (data not shown). These results are in agreement with the fact that weight loss or impaired weight gain are only detected when mice are exposed to doses of TCDD considerably higher than 3 µg/kg body weight (2, 15). Thus, the activity of the CA-AhR seems to correspond to a chronic, relatively low dose exposure to TCDD or other AhR ligands.

[0044] A well-characterized adverse effect of dioxin is involution of the thymus (1). The relative thymus weight of CA-AhR animals was decreased up to six months of age (Fig. 2B). Altered population sizes of single positive CD8⁺ and CD4⁺ T cells have previously been observed in rats exposed to TCDD during gestation (16). This effect was also observed in thymi from new-born CA-AhR mice (data not shown). These results indicate that in the absence of dioxin, CA-AhR mimicked biological effects that are normally elicited by the dioxin-activated form of the AhR.

[0045] The CA-AhR mice showed a significantly reduced life span where only very few homozygous animals survived past an age of 12 months. Several mice were found dead, beginning already at six months of age, most often without any preceding clinical symptoms. Notably, there was a striking sex difference in that male mice died earlier than females. In addition, a difference in the time-course between two independent homozygous lines of mice was also observed (Fig. 2C).

[0046] At necropsy, dramatic stomach lesions were observed in the CA-AhR mice. In contrast to stomachs from wild type mice (Fig. 3A), CA-AhR mice demonstrated grossly visible cysts at 3-4 months of age in the lesser curvature of the stomach (Fig. 3B). The cysts became more numerous with age (Fig. 3C) and in the most severe cases (around 12 months of age), the growths adhered to surrounding organs such as liver, pancreas and abdominal fat (Fig. 3D). In many cases, the stomach wall was thickened throughout the cardia and corpus region of the glandular stomach. The limiting ridge, which defines the border between the forestomach and the glandular part of the rodent stomach (Fig. 3E), was also macroscopically substantially enlarged (data not shown).

[0047] Histopathological analysis revealed glandular structures expanding from the mucosa into the stroma of the limiting ridge, explaining the thickening observed at gross inspection (Fig. 3F). The expansive growth of the cystic glandular structures in the mucosa showed invasion of dysplastic crypts into the submucosa, muscularis propria and eventually into the subserosal region (Fig. 3F, G). In spite of the aggressive behaviour of the invading tumour cells, they retained a remarkably well differentiated appearance after passing through the muscularis mucosa (Fig. 3G). The tumour development progressed over time resulting in a bizarre, distorted tissue architecture observed prior to lethality (12 months of age, Fig. 3H).

[0048] The present inventors also detected glands in the subserosa that were clearly defined by connective tissue and associated with lymphatic tissue, vessels, fat and sometimes nerves (Fig. 3I), indicative of a severe perturbation of the differentiation status of these tissues. These alterations are characteristic for hamartomas of the human stomach. Intestinal metaplasia was common in most cysts of the tumours demonstrating staining of intestinal-type mucous not normally observed in the corpus of the stomach (Fig. 4A-B). Moreover, squamous metaplasia resulting in formation of squamous cysts was also observed (data not shown). A closer analysis of the expansively growing epithelial cells penetrating the muscularis mucosa layer showed that these cells were not surrounded by cells of the muscularis mucosa layer (Figs. 4C-D). This observation rules out herniation as the cause of penetration, consistent with an invasive growth behaviour. The present inventors also detected squamous cysts that were focally located on the caecum and occasionally on the ileum in several CA-AhR mice six months of age or older (Fig. 4E-F). Although the CA-AhR was expressed at the highest level in the glandular part of the stomach with a resulting strong CYP1A1 induction response, the transgene was expressed and functionally active throughout the entire gastrointestinal tract (Fig. 4G). Despite this fact no other major lesions than those described were found in the gastrointestinal tract.

[0049] The gastric tumours were not found in any wild type mice (n>200) but in more than 200 transgenic animals. Moreover, the tumours appeared in three independent lines of CA-AhR mice, indicating that the neoplasia was not an effect of random integration of the expression construct into the genome. Heterozygous mice showed less severe stomach tumours than homozygous mice, indicating a gene-dosage effect (data not shown). Moreover, the severity of the gastric tumours increased with age, and males were more severely affected (data not shown), further illustrating the sex difference in susceptibility to the CA-AhR, previously observed with regard to mortality (Fig. 2C).

[0050] Although the invention has been described with regard to its preferred embodiments, which constitute the best mode presently known to the inventors, it should be understood that various changes and modifications as would be obvious to one having the ordinary skill in this art may be made without departing from the scope of the invention as set forth in the claims appended hereto.

1. Gu, Y. Z., Hogenesch, J. B. & Bradfield, C. A. (2000) Annu. Rev. Pharmacol.Toxicol. 40, 519-561.

2. IARC (1997) LARC Monogr. Eval. Carcinog. Risks Hum. 69, 33-343.

3. Shimizu, Y., Nakatsuru, Y., Ichinose, M., Takahashi, Y., Kume, H., Mimura, J., Fujii-Kuriyama, Y. & Ishikawa, T. (2000) Proc. Natl. Acad. Sci. U S A 97, 779-782.

4. Lahvis, G. P. & Bradfield, C. A. (1998) Biochem. Pharmacol. 56, 781-787.

5. Mimura, J., Yamashita, K., Nakamura, K., Morita, M., Takagi, T. N., Nakao, K., Ema, M., Sogawa, K., Yasuda, M., Katsuki, M. & Fujii-Kuriyama, Y. (1997) Genes Cells 2, 645-654.

6. Lahvis, G. P., Lindell, S. L., Thomas, R. S., McCuskey, R. S., Murphy, C., Glover, E., Bentz, M., Southard, J. & Bradfield, C. A. (2000) Proc. Natl. Acad. Sci. USA 97, 10442-10447.

7. Fernandez-Salguero, P. M., Ward, J. M., Sundberg, J. P. & Gonzalez, F. J. (1997) Vet. Pathol. 34, 605-614.

8. Whitelaw, M. L., Gustafsson, J. Å. & Poellinger, L. (1994) Mol. Cell. Biol. 14, 8343-8355.

9. Bodrug, S. E., Warner, B. J., Bath, M. L., Lindeman, G. J., Harris, A. W. & Adams, J. M. (1994) EMBO J. 13, 2124-2130.

10. Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989) Molecular cloning: A Laboratory Manual. (Cold Spring Harbour Laboratory Press, Cold Spring Harbour, NY).

11. Gradin, K., Toftgård, R., Poellinger, L. & Berghard, A. (1999) J. Biol. Chem. 274, 13511-13518.

12. Mangelsdorf, D. J. & Evans, R. M. (1995) Cell 83, 841-850.

13. Whitelaw, M. L., Göttlicher, M., Gustafsson, J. Å. & Poellinger, L. (1993) EMBO J. 12, 4169-4179.

14. Jenuwein, T. & Grosschedl, R. (1991) Genes. Dev. 5,932-943.

15. Pohjanvirta, R. & Tuomisto, J. (1994) Pharmacol. Rev. 46, 483-549.

16. Gehrs, B. C., Riddle, M. M., Williams, W. C. & Smialowicz, R. J. (1997) Toxicology 122, 229-240.

17. Stemmermann, G. N. (1994) Cancer 74, 556-564.

18. Endoh, Y., Tamura, G., Motoyama, T., Ajioka, Y. & Watanabe, H. (1999) Hum. Pathol. 30, 826-832.

19. Gillner, M., Bergman, J., Cambillau, C., Alexandersson, M., Fernström, B. & Gustafsson, J. A. (1993) Mol Pharmacol 44, 336-345.

20. Kleman, M. I., Övervik, E., Mason, G. G. & Gustafsson, J. Å. (1992) Carcinogenesis 13, 1619-1624.

21. Parkin, D. M., Pisani, P. & Ferlay, J. (1999) Int. J. Cancer 80, 827-841.

22. Stadtländer, C. T. & Waterbor, J. W. (1999) Carcinogenesis 20, 2195-2208.

23. Ekström, A. M., Eriksson, M., Hansson, L. E., Lindgren, A., Signorello, L. B., Nyren, O. & Hardell, L. (1999) Cancer Res. 59, 5932-5937.

24. Svensson, B. G., Mikoczy, Z., Stromberg, U. & Hagmar, L. (1995) Scand. J. Work Environ. Health 21, 106-115.

25. Jain, S., Maltepe, E., Lu, M. M., Simon, C. & Bradfield, C. A. (1998) Mech. Dev. 73, 117-123.

26. Matikainen, T., Perez, G. I., Jurisicova, A., Pru, J. K., Schlezinger, J. J., Ryu, H. Y., Laine, J., Sakai, T., Korsmeyer, S. J., Casper, R. F., Sherr, D. H. & Tilly, J. L. (2001) Nat Genet 28, 355-360.

27. Stewart, H. L., Hare, W. V. & Bennett, J. G. (1953) J. Natl. Cancer Inst. 14, 105-125.

28. Stewart, H. L., Snell, K. C. & Hare, W. V. (1958) J. Natl. Cancer Inst. 21, 999-1019.

29. Allen, J. R. & Norback, D. H. (1973) Science 179, 498-499.

30. Morgan, R. W., Ward, J. M. & Hartman, P. E. (1981) Cancer Res. 41, 5052-5059.

31. Milstone, L. M. & LaVigne, J. F. (1984) J. Invest. Dermatol. 82,532-534.

32. Elizondo, G., Fernandez-Salguero, P., Sheikh, M. S., Kim, G. Y., Fomace, A. J., Lee, K. S. & Gonzalez, F. J. (2000) Mol. Pharmacol. 57, 1056-1063.

33. Ma, Q. & Whitlock, J. P. (1996) Mol. Cell. Biol. 16, 2144-2150.

34. Kolluri, S. K., Weiss, C., Koff, A. & Gottlicher, M. (1999) Genes. Dev. 13, 1742-1753.

35. Ceci, J. D., Kovatch, R. M., Swing, D. A., Jones, J. M., Snow, C. M., Rosenberg, M. P., Jenkins, N. A., Copeland, N. G. & Meisler, M. H. (1991) Oncogene 6, 323-332.

36. Sandmöller, A., Halter, R., Gomez-La-Hoz, E., Gröne, H. J., Suske, G., Paul, D. & Beato, M. (1994) Oncogene 9, 2805-2815.

37. Thompson, J., Epting, T., Schwarzkopf, G., Singhofen, A., Eades-Perner, A. M., van Der Putten, H. & Zimmermann, W. (2000) Int. J. Cancer 86, 863-869.

38. Searle, P. F., Thomas, D. P., Faulkner, K. B. & Tinsley, J. M. (1994) J. Gen. Virol. 75, 1125-1137.

39. Ge, N. L. & Elferink, C. J. (1998) J. Biol. Chem. 273, 22708-22713.

40. Puga, A., Barnes, S. J., Dalton, T. P., Chang, C., Knudsen, E. S. & Maier, M. A. (2000) J. Biol. Chem. 275, 2943-2950.

41. Sharp, R., Babyatsky, M. W., Takagi, H., Tagerud, S., Wang, T. C., Bockman, D. E., Brand, S. J. & Merlino, G. (1995) Development 121, 149-161.

42. Strayer, D. S., Yang, S. & Schwartz, M. S. (1993) Lab. Invest. 69, 660-673.

43. Gaido, K. W., Maness, S. C., Leonard, L. S. & Greenlee, W. F. (1992) J. Biol. Chem. 267, 24591-24595.

Claims

1. An expression construct for a constitutively active dioxin/aryl hydrocarbon receptor (CA-AhR), characterized in that said construct is the mouse AhR sequence having amino acids 288 - 421 deleted.

2. A transgenic non-human animal functionally incorporating an expression construct according to claim 1.

3. The transgenic non-human animal of claim 2, wherein said animal is selected from the group consisting of mice, rats, monkeys, sheep and rabbits.

4. An isolated cell of the transgenic animal of claim 2.

5. An isolated cell line derived from the transgenic animal of claim 2.

6. The cell of claim 4, characterized in that said cell is selected from a germ cell or a somatic cell.

7. An non-human animal model for the study of cancer, comprising a transgenic animal according to claim 2.

8. An non-human animal model for the study of cancer, comprising a transgenic animal according to claim 3.

9. An in vitro model for the study of cancer, comprising a cell according to claim 4.

10. An in vitro model for the study of cancer, comprising a cell line according to claim 5.

11. A method for the screening of drug candidates, characterized in that the anti-carcinogenic effect of said drug candidates is assessed in a non-human transgenic animal according to claims 2 or 3 or a cell thereof or cell line derived therefrom and expressing a constitutively active dioxin/aryl hydrocarbon receptor (CA-AhR).

12. A method for the screening of drug candidates, characterized in that the anti-carcinogenic effect of said drug candidates is assessed in a model according to any one of claims 7 -10.

13. A method for investigating the molecular mechanisms of cancer, characterized in that a model according to any one of claims 7 - 10 is used.

14. A method for investigating the mechanisms of highly differentiated adenocarcinoma in the stomach, characterized in that that a model according to any one of claims 7 - 10 is used.

15. A method of inducing stomach cancer in a non-human animal for research purposes, characterized in that said animal is transfected with a construct according to claim 1.

16. The method according to claim 15, wherein the construct is injected into a fertilised egg of said non-human animal and permitting the egg to develop into an animal containing said construct in its genome.

17. A method of inducing drug metabolising enzymes normally regulated by the Ah-receptor in the presence of a ligand for the study of drug metabolism by any member (-s) of said enzymes, characterized in that a non-human animal is transfected with a construct according to claim 1.

18. A method of inducing drug metabolising enzymes normally regulated by the Ah-receptor in the presence of a ligand for the study of drug metabolism by any member (-s) of said enzymes, characterized in that cultured cells are transfected with a construct according to claim 1.

Ansprüche

1. Expressionskonstrukt für einen konstitutiv aktiven Dioxin/Aryl-Kohlenwasserstoff-Rezeptor (CA-AhR), dadurch gekennzeichnet, dass das Konstrukt die Mäuse-AhR-Sequenz, in der die Aminosäuren 288-421 fehlen, ist.

2. Transgenes nicht-menschliches Tier, das ein Expressionskonstrukt gemäß Anspruch 1 funktionsfähig enthält.

3. Transgenes nicht-menschliches Tier nach Anspruch 2, bei dem das Tier ausgewählt ist aus der Gruppe, die aus Mäusen, Ratten, Affen, Schafen und Kaninchen besteht.

4. Isolierte Zelle des transgenen Tiers nach Anspruch 2.

5. Isolierte Zelllinie, die von dem transgenen Tier nach Anspruch 2 stammt.

6. Zelle nach Anspruch 4, dadurch gekennzeichnet, dass die Zelle aus einer Keimzelle oder einer Körperzelle ausgewählt ist.

7. Nicht-menschliches Tiermodell zum Studium von Krebs, aufweisend ein transgenes Tier nach Anspruch 2.

8. Nicht-menschliches Tiermodell zum Studium von Krebs, aufweisend ein transgenes Tier nach Anspruch 3.

9. In vitro-Modell zum Studium von Krebs, aufweisend eine Zelle nach Anspruch 4.

10. In vitro-Modell zum Studium von Krebs, aufweisend eine Zelllinie nach Anspruch 5.

11. Verfahren zum Durchmustern von Arzneimittel-Anwärtern, dadurch gekennzeichnet, dass die anti-karzinogene Wirkung der Arzneimittel-Anwärter in einem nicht-menschlichen transgenen Tier nach Anspruch 2 oder 3 oder einer Zelle davon oder einer davon stammenden Zelllinie, das (die) einen konstitutiv aktiven Dioxin/Aryl-Kohlenwasserstoff-Rezeptor (CA-AhR) exprimiert, geprüft wird.

12. Verfahren zum Durchmustern von Arzneimittel-Anwärtern, dadurch gekennzeichnet, dass die anti-karzinogene Wirkung der Arzneimittel-Anwärter in einem Modell gemäß einem der Ansprüche 7 - 10 geprüft wird.

13. Verfahren zum Untersuchen der molekularen Mechanismen von Krebs, dadurch gekennzeichnet, dass ein Modell gemäß einem der Ansprüche 7 - 10 verwendet wird.

14. Verfahren zum Untersuchen der Mechanismen hochgradig differenzierter Ardenokarzinome im Magen, dadurch gekennzeichnet, dass ein Modell gemäß einem der Ansprüche 7 - 10 verwendet wird.

15. Verfahren zum Auslösen von Magenkrebs bei einem nicht-menschlichen Tier für Forschungszwecke, dadurch gekennzeichnet, dass das Tier mit einem Konstrukt gemäß Anspruch 1 transfiziert wird.

16. Verfahren nach Anspruch 15, bei dem das Konstrukt in ein befruchtetes Ei des nicht-menschlichen Tiers injiziert wird und dem Ei erlaubt wird, sich zu einem Tier zu entwickeln, das das Konstrukt in seinem Genom enthält.

17. Verfahren zum Erzeugen von Arzneimittel metabolisierenden Enzymen, die normalerweise durch den Ah-Rezeptor in Anwesenheit eines Liganden reguliert werden, zum Studium des Arzneimittel-Metabolismus durch irgendein Mitglied (irgendwelche Mitglieder) der Enzyme, dadurch gekennzeichnet, dass ein nicht-menschliches Tier mit einem Konstrukt gemäß Anspruch 1 transfiziert wird.

18. Verfahren zum Erzeugen von Arzneimittel metabolisierenden Enzymen, die normalerweise durch den Ah-Rezeptor in Anwesenheit eines Liganden reguliert werden, zum Studium des Arzneimittel-Metabolismus durch irgendein Mitglied (irgendwelche Mitglieder) der Enzyme, dadurch gekennzeichnet, dass kultivierte Zellen mit einem Konstrukt gemäß Anspruch 1 transfiziert werden.

Revendications

1. Construction d'expression d'un récepteur hydrocarbone dioxine aryle constitutivement actif (CA-AhR), caractérisée en ce que ladite construction est la séquence AhR de souris ayant une délétion des acides aminés 288 à 421.

2. Animal transgénique non-humain incorporant de façon fonctionnelle une construction d'expression selon la revendication 1.

3. Animal transgénique non-humain selon la revendication 2, choisi dans le groupe constitué des souris, rats, singes, chèvres et lapins.

4. Cellule isolée de l'animal transgénique selon la revendication 2.

5. Lignée cellulaire isolée dérivée de l'animal transgénique selon la revendication 2.

6. Cellule selon la revendication 4, caractérisé en ce que ladite cellule est choisie parmi une cellule germinale et une cellule somatique.

7. Modèle animal non-humain pour l'étude du cancer, comprenant un animal transgénique selon la revendication 2.

8. Modèle animal non-humain pour l'étude du cancer, comprenant un animal transgénique selon la revendication 3.

9. Modèle in vitro pour l'étude du cancer, comprenant une cellule selon la revendication 4.

10. Modèle in vitro pour l'étude du cancer, comprenant une lignée cellulaire selon la revendication 5.

11. Procédé de criblage de candidats-drogue, caractérisé en ce que les effets anti-carcinogènes desdits candidats-drogue sont testés chez un animal transgénique non-humain selon la revendication 2 ou 3, ou une cellule de cet animal transgénique ou une lignée cellulaire dérivée de ceux-ci, exprimant un récepteur hydrocarbone dioxine aryle constitutivement actif (CA-AhR).

12. Procédé de criblage de candidats-drogue, caractérisé en ce que les effets anti-carcinogènes desdits candidats-drogue sont évalués dans un modèle selon l'une quelconque des revendications 7 à 10.

13. Procédé pour étudier les mécanismes moléculaires du cancer, caractérisé en ce qu'on utilise le modèle selon l'une quelconque des revendications 7 à 10.

14. Procédé pour étudier les mécanismes des adénocarcinomes hautement différenciés dans l'estomac, caractérisé en ce qu'on utilise un modèle selon l'une quelconque de revendications 7 à 10.

15. Procédé d'induction d'un cancer stomacal chez un animal non-humain à but de recherche, caractérisé en ce que ledit animal est transfecté par une construction selon la revendication 1.

16. Procédé selon la revendication 15, dans lequel la construction est injectée dans un oeuf fertilisé dudit animal non-humain, permettant à l'oeuf de se développer dans un animal contenant ladite construction dans son génome.

17. Procédé d'induction d'enzymes métabolisant une drogue, normalement régulés par le récepteur Ah, en présence d'un ligand pour l'étude du métabolisme de la drogue par n'importe quel membre desdits enzymes, caractérisé en ce qu'un animal non-humain est transfecté par une construction selon la revendication 1.

18. Procédé d'induction d'enzymes métabolisant une drogue, normalement régulés par le récepteur Ah, en présence d'un ligand pour l'étude du métabolisme de la drogue par n'importe quel membre desdits enzymes, caractérisé en ce que les cellules cultivées sont transfectées par une construction selon la revendication 1.

Drawing